CN113398250B

CN113398250B - Liquid gonadotrophin medicinal preparation and use

Info

Publication number: CN113398250B
Application number: CN202110585922.3A
Authority: CN
Inventors: 海伦·乌尔丽卡·格伦; 夏洛特·霍耶·佩德森
Original assignee: Ferring BV
Current assignee: Ferring BV
Priority date: 2016-02-24
Filing date: 2017-02-24
Publication date: 2024-02-23
Anticipated expiration: 2037-02-24
Also published as: KR20180114178A; ES2966262T3; TW201733612A; US20230372447A1; EP4257149A3; ES2862193T3; PT3791860T; MA52595B1; MY193138A; US20190060409A1; TN2018000284A1; TWI755377B; DK3791860T5; CL2018001984A1; HRP20231477T1; MA43686A; LT3791860T; DK3791860T3; IL260626A; CN108883061B

Abstract

The present invention relates generally to the field of stabilization of gonadotrophin formulations, in particular liquid gonadotrophin pharmaceutical formulations and uses. A liquid gonadotrophin pharmaceutical formulation consisting of the following components: gonadotropin, 50-160mM arginine HCl, 0.05 to 1.5mg/ml L-methionine, 0.001-0.05mg/ml polysorbate 20, 4.0 to 6.0mg/ml phenol, pH 6.0 to 7.5 and water for injection (WFI), which are capable of achieving stabilization.

Description

Liquid gonadotrophin medicinal preparation and use

Technical Field

The present invention relates generally to the field of stabilization of gonadotrophin preparations, in particular liquid gonadotrophin pharmaceutical preparations and uses. A liquid gonadotrophin pharmaceutical formulation consisting of the following components: gonadotropin, 50-160mM arginine HCl, 0.05 to 1.5mg/ml L-methionine, 0.001-0.05mg/ml polysorbate 20, 4.0 to 6.0mg/ml phenol, pH 6.0 to 7.5 and water for injection (WFI), which are capable of achieving stabilization.

Background

Gonadotropins are a class of hormones that are essentially involved primarily in the fertility cycle of females and males. Gonadotropins can be extracted from urine for research and therapeutic purposes, however, several gonadotropins like e.g. hCG, LH and FSH can also be recombinantly produced.

In particular, gonadotrophin is useful in the treatment of infertility.

All four major gonadotrophins belong to the same glycoprotein family. These are Follicle Stimulating Hormone (FSH), thyroid Stimulating Hormone (TSH), luteinizing Hormone (LH) and (human) chorionic gonadotropin (hCG). All of these gonadotrophins are heterodimers and consist of alpha and beta subunits; the alpha subunit is common to all gonadotrophins, i.e. the alpha subunit is the same for all four gonadotrophin and the beta subunit is different respectively. The actions of FSH are mediated by different FSH receptors. The β chains of LH and HCG have 82% protein sequence homology and exert their effects through the same LH receptor.

FSH is naturally secreted by the anterior pituitary and has the function of supporting follicular development and ovulation. FSH comprises 92 amino acid alpha subunits, also common to other glycoprotein hormones, e.g. LH and hCG, and the unique 111 amino acid beta subunits of FSH, which confer biological specificity to the hormone (Pierce and Parsons,1981,Glycoprotein hormones:structure and function,Ann Rev Biochem,50:465-495). The mature β subunit of hCG consists of 145 amino acids. Each subunit of FSH and hCG is post-translationally modified by the addition of a complex carbohydrate residue. For FSH, both subunits bear two N-linked glycan attachment sites, the alpha subunit of amino acids 52 and 78 and the beta subunit of amino acid residues 7 and 24 (Rathnam and Saxena, (1975) Primary amino acid sequence of follicle stimulating hormone from human pituitary glands. I.alpha. Subsubuit, J Biol chem.250 (17): 6735-6746;Saxena and Rathnam, (1976) Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands, J Biol chem.251 (4): 993-1005)). Thus, FSH was glycosylated to about 30% mass (Dias and Van Roey, (2001) Structural biology of human follitropin and its receptor. Arch Med Res.32 (6): 510-519; fox et al (2001) Three-dimensional structure of human follicle-stilling horone. Mol endocrinol.15 (3), 379-89). The beta subunit of hCG comprises N-and O-glycosylation (N-13, N-30, O-121, O127, O-132 and O-138). The extra glycosylation in the β subunit of hCG makes it more hydrophilic than FSH. The β -subunit provides specificity for receptor interactions.

Urinary gonadotropins have been used clinically for over 40 years and their safety has been well established. Over time, a new generation of Highly Purified (HP) urinary gonadotropins has been introduced as compared to the first generation. Higher purity is obtained by adding additional purification steps, such as anion exchange and hydrophobic interaction chromatography steps, to remove urine proteins without FSH and/or LH bioactivity. The significant increase in purity of the new generation gonadotrophin formulations facilitates a more comprehensive characterization study that provides more information to the composition.

Both purified urinary FSH and human menopausal gonadotropin (hMG) are isolated from urine of postmenopausal women and have been used for many years in infertility treatment to induce (mono) ovulation or to stimulate multiple follicles in patients receiving Controlled Ovarian Stimulation (COS) prior to Assisted Reproductive Technology (ART). FSH is a polypeptide of the FSH type,(follitropin. Alpha., merck Serono) and +.>Two recombinant versions of (follitropin β, merck) were marketed in the middle of 90 s of the 20 th century. Both products were expressed in Chinese Hamster Ovary (CHO) cell lines (Howles, c.m. (1996)), genetic engineering of human FSH (Gonal-f), hum reprod.

CHO cells are commonly used for the production of pharmaceutical recombinant proteins. Structural analysis showed sialic acid was only linked by a 2, 3-linkage. Many human glycoproteins contain a mixture of α2, 3-and α2, 6-linkages of sialic acid residues. Thus, recombinant proteins expressed using CHO systems differ from their natural counterparts in the type of their terminal sialic acid linkages.

Infertility syndrome

In the context of the present invention, "infertility" shall be defined as reduced ability or inability to conception and offspring of fertility. Women who are able to become pregnant but who later undergo multiple abortions are also considered to be infertility. Infertility is also specifically defined as failure to become pregnant after one year of normal intercourse without contraception. Infertility can be caused by a variety of causes. Studies have shown that slightly more than half of infertility cases are caused by female conditions. The remaining cases are caused by sperm disease and unknown causes. There are several possibilities for treating infertility.

These possibilities include timed intercourse, medical treatment of endometriosis, ovulation Induction (OI), fibroids and Female Sexual Dysfunction (FSD) using Assisted Reproductive Technology (ARTs), and surgery to correct abnormalities.

In assisted reproductive technologies and OI, drugs that stimulate ovulation are used. Next to this, FSH, which is mainly responsible for ovarian stimulation, gonadotrophin preparations may also contain LH and/or hCG.

Currently, in clinical practice, several different pharmaceutical products containing gonadotrophin derived from urine of pregnant or postmenopausal women are used for the treatment of infertility, such as HMG (human menopausal gonadotrophin) formulations containing 1:1 FSH and LH bioactivity (see e.g. USP 35 th edition, gonadotrophin monograph), and formulations containing FSH bioactivity only. Gonadotrophin products produced by recombinant DNA technology have been marketed since 1995.

It is therefore important to provide stable formulations of gonadotropin compounds, alone or in combination.

It is therefore an object of the present invention to provide formulations, in particular liquid formulations, of one or more gonadotropins, in particular formulations comprising hCG, optionally in combination with FSH, which are stable. It is another object of the present invention to provide a method of stabilizing the formulation. It is a further object to provide such a formulation which is stable for 12 months, preferably 24 months, more preferably 24 months under storage conditions plus 1 month "in use" (i.e. at room temperature).

Disclosure of Invention

The present invention relates to stable liquid gonadotrophin formulations. In a preferred embodiment, the formulation comprises hCG. In another preferred embodiment, the formulation comprises FSH and hCG. The gonadotropins in the formulations of the present invention are preferably urine-or plasma-derived, but in an alternative embodiment may be recombinantly produced.

Hereinafter, the term "hMG" is used interchangeably with "urinary gonadotropin". In most cases, gonadotropins in urine come from human urine.

Human chorionic gonadotropin (hCG) contributes to LH (luteinizing hormone) activity, which is why currently approved drugs refer to indications. This is a well known fact and is described as part of SmPCs of hMG formulations, e.g-a product, which is authorized for the same indication as presently claimed.In these SmPCs-FSH and LH activities are included, but it has additionally been demonstrated that hCG provides at least part of the activity for LH. Thus, any reference to hCG in the context of the present invention includes formulations comprising LH activity attributable to hCG.

Preferred embodiments include the following:

1. a liquid gonadotrophin pharmaceutical formulation comprising gonadotrophin, arginine in an amount of 50 to 160mM, and methionine in an amount of 0.05 to 1.5mg/ml, wherein said formulation does not comprise additional buffering agents, and wherein the pH of said formulation is between 6.0 and 7.5.

2. The pharmaceutical formulation according to item 1, wherein the gonadotropin comprises hCG (human chorionic gonadotrophin), and optionally FSH and/or LH.

3. The pharmaceutical formulation of item 1 or 2, wherein the gonadotrophin comprises hMG (human menopausal gonadotrophin).

4. A pharmaceutical formulation according to any one of items 1 to 3, wherein the gonadotropins (e.g. FSH, LH and/or hCG) are of human and urinary origin.

5. A pharmaceutical formulation according to any one of items 1 to 3, wherein the gonadotropins (e.g. FSH, LH and/or hCG) are recombinant.

6. The pharmaceutical formulation according to any of the preceding items, further comprising a preservative, preferably phenol.

7. The pharmaceutical formulation according to any one of the preceding items, further comprising a surfactant, preferably polysorbate, even more preferably polysorbate 20.

8. The pharmaceutical formulation according to item 6 or 7, wherein the preservative, preferably phenol, is present in an amount of 4-6mg/ml, preferably in an amount of 5 mg/ml.

9. The pharmaceutical formulation according to item 7 or 8, wherein the surfactant, preferably polysorbate 20, is present in an amount of 0.001-0.05mg/ml, preferably in an amount of 0.005 mg/ml.

10. The pharmaceutical formulation of any one of the preceding items, wherein the arginine is preferably L-arginine HCl.

11. The pharmaceutical formulation according to any of the preceding items, wherein hMG is present in an amount of 300-900IU/ml, more preferably in an amount of 500-700IU/ml, even more preferably in an amount of 600-650IU/ml, very preferably in an amount of 625 IU/ml.

12. The pharmaceutical formulation according to any one of the preceding items, consisting of the following components:

-625IU/ml hMG

0.15mg/ml methionine

-150mM arginine

5mg/ml phenol

-0.005mg/ml polysorbate 20

Water for injection (WFI), and

-wherein the pH of the formulation is 6.8+/-0.3.

13. The liquid pharmaceutical formulation according to any one of the preceding claims for use in a method of treating infertility.

14. The pharmaceutical formulation for use according to item 13, wherein the treatment is treatment of Ovulation Induction (OI), assisted Reproductive Technology (ART) and/or male hypogonadism.

15. A method for stabilizing a liquid pharmaceutical formulation comprising hMG, comprising the steps of:

-providing a human female urine sample,

-the extraction of hMG,

mixing said extract with arginine and methionine in amounts defined in any of the foregoing,

optionally further adding phenol and polysorbate in amounts as defined in any of the preceding claims,

Adjusting the pH to provide a formulation having a pH between 6.0 and 7.5,

wherein no further buffer is added.

Gonadotropins, e.g., FSH and hCG, and LH are useful in the treatment of infertility, as described above. In this respect, it is evident that the liquid formulations of these gonadotrophins may be unstable; even those intended for single use. Instability is even more pronounced if the liquid formulation contains a preservative, such as that prescribed for all multi-dose formulations.

The formulations of the present invention may be used for single use or multiple use, respectively.

The FSH formulated in the formulation of the invention may be urine or plasma derived or recombinant FSH (rFSH). In a preferred embodiment, the FSH is urinary FSH or rFSH; particularly preferred is urinary FSH.

As mentioned above, gonadotropins such as FSH, LH or hCG can now be recombinantly produced. Thus, unless otherwise indicated, references to gonadotrophin herein generally always include both those derived from urine or plasma as well as recombinant (r) gonadotrophin. Thus, for example, reference to "FSH" also includes rFSH. The production of FSH, hCG and LH and the amino acid and nucleic acid sequences are well known to those skilled in the art.

The sequences that can be used in the context of the present invention are as follows:

FSH, LH and hCG alpha subunit (SEQ ID NO: 1)

(see also Fiddes, J.C.and Goodman, H.M.the gene encoding the common alpha subunit of the four human glycoprotein hormones J.mol.appl.Genet.1 (1), 3-18 (1981))

MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQ

CMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS (leader peptide is underlined) (116)

FSH beta subunit (SEQ ID NO: 2)

(see also Saxena, B.B.and Rathnam, P.Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands J.biol.chem.251 (4), 993-1005 (1976))

MKTLQFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDPARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPSYCSFGEMKE (leader peptide is underlined) (129)

hCG beta subunit (SEQ ID NO: 3)

(see alsoFiddes,JC,Goodman HM.The cDNA for the beta-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3'-untranslated region Nature.1980Aug 14；286(5774):684-7)

MEMFQGLLLLLLLSMGGTWASKEPLRPRCR PINATLAVEKEGCPVCITVN TTICAGYCPT MTRVLQGVLP ALPQVVCNYR DVRFESIRLPGCPRGVNPVV SYAVALSCQC ALCRRSTTDC GGPKDHPLTC DDPRFQDSSSSKAPPPSLPS PSRLPGPSDT PILPQ (leader peptide is underlined) (165)

LH beta subunit (SEQ ID NO: 4)

(see also Sairam, M.R.and Li, C.H.human pituitary lutropin. Isolation, properties, and the complete amino acid sequence of the beta-subsuit Biochim. Biophys acta 412 (1), 70-81 (1975))

MEMLQGLLLLLLLSMGGAWASREPLRPWCH

PINAILAVEKEGCPVCITVN

TTICAGYCPTMMRVLQAVLPPLPQVVCTYRDVRFESIRLPGCPRGVDPVVSFPVALSCRCGPCRRSTSDC GGPKDHPLTC DHPQLSGLLFL (leader peptide is underlined) (141)

In another embodiment, the rFSH or rhhcg of all embodiments is long-acting rFSH or rHCG, respectively. Such long-acting FSH preparations may be obtained, as is generally known to the person skilled in the art, for example by modifying FSH molecules or by modifying the preparation.

Thus, "FSH" as used herein includes all possible urine-derived or recombinant forms of FSH as described above as well as all possible combinations of FSH forms. Also included are single use formulations and further formulations of one or more of the same or different gonadotropins for use in multiple doses.

One possible product may be a formulation comprising FSH (in a preferred embodiment hCG, and/or optionally LH, LH activity, etc.), all in different containers. LH activity (if present) may be derived from LH or hCG. LH can be replaced with an equivalent dose of hCG and vice versa; the "equivalent dose" in the context of the present invention may be calculated as known in the art.

A particularly preferred gonadotropin combination is FSH and hCG, preferably as hMG formulation in one container, but optionally also e.g. in a different container, such as a vial or cartridge.

Possible combinations that may also be provided in different containers also include: urine (u) FSH and uhCG or uhsh and uLH; further (rhCG or rLH or rFSH) and (uhCG or uhh or rhCG or rLH) and all possible permutations thereof. In a very preferred embodiment, the formulation of the invention comprises FSH and hCG. In another equally preferred embodiment, the formulation of the invention comprises hCG.

The gonadotrophin formulation of the present invention is a liquid formulation. Preferably, the formulation is injectable. The formulations may be provided as a product having one, two or more pharmaceutical compositions comprising FSH or FSH/hCG, and/or LH for administration alone or together. If administered alone, they may be administered sequentially. The product may be provided in any suitable package. For example, the product may comprise a plurality of pre-filled syringes, each syringe comprising FSH (FSH composition), or further hCG (hCG composition), for example, wherein the syringes may be packaged in blister packs or other devices to maintain sterility. The product may optionally contain instructions for using the gonadotrophin formulation.

According to a further aspect, the gonadotrophin formulation of the present invention is provided as a multi-dose formulation. However, the invention also relates specifically to formulations intended for single use. The invention also relates to the stabilization of a formulation as part of a kit. Such a kit comprises at least one container containing one or more daily doses of gonadotropins, e.g. FSH, or e.g. two containers (e.g. vials), each container containing a different gonadotropin, e.g. hCG, together with e.g. further instructions (e.g. administration) and e.g. further injection methods. In a preferred embodiment, an injection pen for multiple injections is used, whereby gonadotrophin solutions are filled in respective cartridges. The active ingredients may be in different cartridges, but of course may be injected simultaneously, or sequentially, as is well known to those skilled in the art. Furthermore, two or more active ingredients may be in the same cartridge.

In a very preferred embodiment, the formulation of the invention is for parenteral use, even more preferably for subcutaneous injection.

In a preferred embodiment, the hMG is present in the formulation in an amount of 35-850IU/ml, preferably 50-800IU/ml, even more preferably 100-700IU/ml, most preferably 625IU/ml, typically in a multi-dose formulation.

A particularly preferred formulation for excipients comprising a multi-dose formulation of hMG as described above, and/or comprising hMG, and/or hCG, and/or all other gonadotrophin according to the invention, has the following composition:

50-160mM arginine HCl, preferably 150mM arginine HCl

0.05 to 1.5mg/ml, preferably 0.15mg/ml, L-methionine

0.001-0.05mg/ml, preferably 0.005mg/ml, polysorbate 20

4.0 to 6.0mg/ml, preferably 5.0mg/ml, phenol

pH 6.0 to 7.5, preferably pH 6.8+/-0.3, (pH refers to the pH of the entire solution)

WFI。

Typical concentrations of the active ingredient of the preparation comprising recombinant hCG and/or FSH are as follows, although the concentration of the active ingredient does not have any effect on the performance of the invention:

for rFSH:30-150 mug/ml

For rhCG:5-200 mug/ml

The preferred excipients for such recombinant formulations are the same as for the multi-dose hMG formulations described above. Typical single dose formulations are also included in the present invention, as described above, except that they do not contain a preservative such as phenol.

Injectable depot forms can be prepared by forming microencapsulated matrices of gonadotrophin (and other agents, if present) in biodegradable polymers. The polymer-based depot forms/slow release systems may be, for example, micro-or nano-particles, hydrogels, micelles, emulsions or implants, depending on their chemical nature. The rate of gonadotropin release can be controlled depending on the ratio of gonadotropin to polymer and the nature of the particular polymer used. Examples of biodegradable polymers include polylactide/polyglycolide copolymer systems, polyvinylpyrrolidone, poly (orthoesters), poly (anhydrides), poly (ethyleneglycol), polyamino acids, polysaccharides such as sodium hyaluronate (NaHA) or other salts, gelatin, chitosan, and the like. All mentioned polymers may be derivatized or modified to optimize protein drug delivery or stability thereof. Depot injectable formulations are also prepared by entrapping gonadotrophin in a lipid system or polymeric lipid mixture that is compatible with body tissue as micelles, liposomes or microemulsions.

The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter and/or by incorporating sterilizing agents. Sterile solid compositions that are soluble or dispersible in sterile water or other sterile injection medium may be formed prior to use. The injectable formulation may be provided in any suitable container, such as vials, prefilled syringes, injection cartridges, and the like, as previously described.

The pH and precise concentrations of the various components used as formulations of the pharmaceutical compositions of the present invention are primarily adjusted in accordance with conventional practice in the art, see, for example, textbooks on medicine, fifth edition, edited by John P.Griffin and John O' Grady. In a preferred embodiment, the composition of the invention is provided as a parenterally administered composition. General methods of preparing parenteral formulations are known in the art and are described on pages 780-820 of REMINGTON, written on pharmaceutical science and practice. The parenteral compositions may be provided in liquid formulations or as solids that will be mixed with a sterile injectable medium prior to administration. In a particularly preferred embodiment, the parenteral compositions are provided in dosage unit form for ease of administration and uniformity of dosage.

FSH, hCG and/or LH formulated according to the present invention may be obtained from urine by conventional methods well known in the art or may be recombinantly produced. For a possible production method, reference is further made to, for example, patent WO 2009/127826.

A preferred embodiment of the invention is the presently described formulation comprising hCG.

hCG may be obtained by any method known in the art. The hCG used in the present invention includes urinary-derived (urinary-derived) hCG and recombinant hCG. Particularly preferred are formulations comprising hCG of urinary origin. Also included is a formulation derived at least in part from hCG having LH activity (in other words hCG is the molecule responsible for the LH activity). hCG of human origin may be purified from any suitable source (e.g. urine and/or placenta) by any method known in the art. Methods for expressing and purifying recombinant hCG are well known in the art.

LH can be obtained by any method known in the art. The LH used in the present invention includes human-derived (LH) and recombinant LH. LH of human origin can be purified from any suitable source (e.g., urine) by any method known in the art. Methods for expression and purification of recombinant LH are known in the art.

The term "pharmaceutical composition" is used interchangeably herein with "pharmaceutical formulation".

The stable pharmaceutical composition of the present invention can be used for treating infertility. "treating infertility" in the context of the present invention includes treating infertility by methods that control ovarian (super) stimulation (COS) or include steps or stages of ovarian stimulationPregnancy disorders such as intrauterine insemination (IUI), in Vitro Fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The term also includes Ovulation Induction (OI) or a method comprising an ovulation induction step or phase. The term also includes treating infertility in a subject suffering from infertility from fallopian tube or infertility from unknown causes, including treating a subject suffering from endometriosis, such as endometriosis stage I or II (as defined by the American Society of Reproductive Medicine (ASRM) for the phase-by-phase classification of endometriosis system, revised american society of reproductive medicine endometriosis: 1996, fertility and infertility, 1997; 67, 817-821), and/or treating infertility in a companion subject suffering from infertility from male factor. The term preferably includes use in Assisted Reproductive Technologies (ARTs), ovulation Induction (OI) or intrauterine insemination (IUI), for example. The pharmaceutical composition may be used in medical indications, for example using known FSH preparations or FSH and hCG preparations. In a typical embodiment, the formulation of the invention is used in combination with European approval The same medical indications as indicated in the following in one exemplary case:

treating female and male infertility:

-anovulatory women: the MENOPUR can be used to stimulate follicular development in amenorrhea patients. Clomiphene (or a similar ovulatory agent affecting the steroid feedback mechanism) is the treatment of choice in women with various menstrual cycle disorders, including anovulatory cycle and luteal phase insufficiency of normal prolactin, and amenorrhea patients with evidence of endogenous estrogen production but normal prolactin and gonadotrophin levels. Non-responders may then be selected for treatment with urotropin.

-women receiving superovulation in a medical assisted fertilization program: the mecofur may be used to induce multiple follicular development in a patient undergoing assisted conception techniques such as In Vitro Fertilization (IVF).

-male hypogonadism: the MENOPUR can be used in combination with human chorionic gonadotrophin (such as chord) to stimulate spermatogenesis.

Or approved in the United statesThe indications of (2) are as follows:

as part of the Assisted Reproductive Technology (ART) cycle, multiple follicles develop and gestate in ovulatory women.

Or,can be described as follows:

MENOPUR refers to infertility treatment in the following clinical situations:

anovulation, including polycystic ovarian disease (PCOD), is found in women who are not effective in clomiphene treatment.

Controlled superovulation of multiple follicular development is induced for Assisted Reproductive Technologies (ART), such as in vitro fertilization/embryo transfer (IVF/ET), gamete intrafallopian transfer (GIFT) and intracytoplasmic sperm injection (ICSI).

Stimulation of follicular development in women suffering from hypogonadism with hypogonadotrophin secretion.

The present invention also provides the use of a stabilized gonadotrophin formulation as described herein (according to aspects of the present invention) in the treatment of infertility or in the manufacture of a medicament for the treatment of infertility.

In a preferred embodiment, the formulation of the invention is used for ovulation induction, assisted Reproductive Technology (ART) and/or for hypogonadism in male gonadotrophs.

The pharmaceutical compositions may be further formulated as well known compositions for any route of administration, such as oral, rectal, parenteral, transdermal (e.g. patch technology), intravenous, intramuscular, subcutaneous, intracisternal, intravaginal, intraperitoneal, topical (powder, ointment or drops) or as an oral or nasal spray. Typical compositions comprise a pharmaceutically acceptable carrier, such as an aqueous solution, non-toxic excipients, including salts and preservatives, buffers, and the like, as described in Remington, fifteenth edition of pharmaceutical science (Matt Publishing Company, 1975), pages 1405-1412 and 1461-87; and USP-NF, the fourteenth edition of national formulary XIV (American society of medicine, 1975), and the like. The present invention, in some embodiments, relates to a specific provision of a formulation that exhibits surprisingly high stability and other advantages, such as no unacceptable coloration, no unacceptable turbidity, reduced or no pain upon injection, reduced or no skin irritation upon injection. This was only achieved by the findings of the present invention, which suggest that the specific composition makes possible the advantageous properties and high stability of the liquid gonadotrophin formulations according to the present invention.

Although gonadotrophin formulations have been on the market for decades, it is clear to formulation scientists that the formulation of these proteins is associated with a number of difficulties. These difficulties exist and vary greatly based on a number of factors, such as

The fact that the proteins are being formulated (the proteins themselves are in any case very difficult to formulate)

The fact that the protein is specifically glycosylated (glycosylation may be affected by the specific choice of manufacture and excipients)

The particular proteins formulated (formulation chemistry varies significantly depending on the actual protein: for example it has been shown that even closely related proteins like FSH and hCG behave differently in the same formulation, see for example WO 2012/0137842)

Whether the protein is naturally obtained, e.g. urine, or recombinantly produced

Whether or not a preservative is required (some of which achieve their intended purpose of preventing microbial growth, but have a negative effect on the stability of the final formulation, this is true for all meta-cresols, phenol and benzyl alcohol (with or without benzalkonium chloride), which are currently the only preservatives approved for use with gonadotrophin; furthermore, preservatives can have a negative effect on color, depending on the other excipients and active ingredients contained in the formulation)

The particular surfactants used (these may in some cases lead to turbidity, also depending on the other excipients contained in the respective formulations)

The particular buffer used (e.g. citrate buffers often cause pain and skin irritation when injected)

Particular excipients for stabilization, which will stabilize the different compositions in an unpredictable manner.

Depending on the excipient used for stabilization, it will stabilize the different compositions in an unpredictable way.

Thus, formulation scientists face a number of problems on the one hand and a number of possible excipients on the other hand, resulting in the complex problem of providing formulations with good stability on the one hand and no coloration or turbidity or pain on injection on the other hand.

Thus, it is very surprising that within the pH range of the present invention, the arginine and methionine combination of the present invention solves these particular problems without the addition of a buffer.

In this regard, "no (added/additional) buffer" and "no buffer included" are synonymously used in this application. The expression shall mean the absence/absence of other compounds in the formulation which are considered to have buffering capacity. A buffer solution is said to be if the solution resists changes in ionic activity when substances are added that are expected to change ionic activity. A buffer is a substance or combination of substances that imparts such resistance to a solution. Buffer solutions are systems in which ions are in equilibrium with a substance capable of removing or releasing ions. It refers to the amount of material that can be added to a solution without causing a significant change in ionic activity. It is defined as the ratio of added acid or base (expressed in gram-equivalents/liter) to the change in pH units. The capacity of the buffer solution is typically adjusted to the conditions of use by adjusting the concentration of the buffer substance (USP NF). Buffer capacity is typically expressed as the number of equivalents (Eq) of a strong acid (e.g., HCl) or a strong base (e.g., naOH) that results in a unit pH change of one liter of the solution (preferably at one atmosphere and 21 ℃) (Skoog West and Holler, analytical chemistry basis, fifth edition). One equivalent of HCl equals 1 mole of HCl and one equivalent of NaOH equals 1 mole of NaOH. In the present invention, the buffer capacity will be expressed as the number of equivalents (Eq) of strong acid (e.g. HCl) or strong base (e.g. NaOH) that cause a unit pH change of one liter of the solution. Thus, according to the present invention, the formulation should not contain additional buffering agents that contribute ≡0.5 mEq/(liter. Times. PH), preferably at 1atm,21 ℃, within the pH range disclosed for the formulation of the present invention.

Method for determining and calculating buffer capacity

The buffer capacity can be determined and calculated as follows.

A volume of the solution to be tested is titrated with an acid (e.g., HCl) or a base (e.g., naOH). Appropriate concentrations of acid and base, e.g., 0.2N, should be used to perform a sufficiently accurate titration.

Titration was performed by adding a small amount of HCl or NaOH to the test solution. For each addition, the volume added and the corresponding pH were recorded.

The volumes of accumulated acid and base are plotted against the measured pH, see for example figure 1. Performing linear least squares regression fit on the relevant pH region, and calculating R ² To confirm the validity of the fit line.

The buffer capacity in mEq/(liter. Times.pH units) was calculated by a linear regression method.

Equation 1

Wherein,

x = buffer capacity [ μL 0.2N HCl/NaOH with pH shifted by 1 pH unit ]

Y-b=1 PH unit

α = slope;

and

equation 2

Where mEq is the milliequivalent of the acid or base given by the concentration, e.g., 0.2N HCl/NaOH, equal to 0.2 equivalents/L HCl/NaOH, equal to 200mEq/L HCl/NaOH.

x=buffer capacity [ mEq/liter×pH Unit ]

Y-b=1 pH unit

c = concentration of acid or base, e.g. for

V = volume of the solution

10 ⁶ Is a conversion factor from mu L to L

Using equation 2 and batch C-01 _{pH range 6.564-6.947} From table 9:

to determine whether a component will contribute to a buffer capacity of ≡0.5 mEq/(liter. Times.pH units), the buffer capacity was determined as described above for both the solution containing the component and the same solution without the component. The difference in buffer capacity determined by the two solutions gives the contribution of a given component to the buffer capacity.

The terms "no (added/additional) buffer" and "no buffer contained"Also meansThe formulations of the present invention do not contain any of the following buffers (FDA approved buffers for parenteral use at a pH in the range of 6-7.5):

histidine

Phosphate salts

Citrate salt

Tromethamine (Tris)

Hydroxyethyl piperazine ethane sulfonic acid (HEPES)

Carbonate salt

In addition, these terms are also meant to exclude any other FDA approved parenterally-used buffers, particularly

Acetate salt

Adipic acid

Ammonium sulfate

Succinate salt

Asparagine derivatives

Aspartic acid

Glutamate (glutamic acid)

Glycine (Gly)

Lactate salt

Lysine

Maleic acid salt (maleic acid)

Fumarate (fumaric acid)

Malate salt

Meglumine (meglumine)

Propionate salts

Alanine (Ala)

Phenylalanine (Phe)

Cysteine (S)

Isoleucine (Ile)

Leucine (leucine)

Proline (proline)

Serine (serine)

Tartrate salt

Threonine (Thr)

Tryptophan

Tyrosine

Valine (valine)

Without the addition of buffer, it is generally assumed that the pH of the final solution cannot be easily maintained within the desired range (and that it is difficult to achieve a specific target pH during adjustment) (in the present invention, the pH should be between 6.0 and 7.5, more preferably between 6.5 and 7.4, preferably 6.8 or about 6.8), but will fluctuate drastically.

It is important to maintain the accurate pH of the drug product. The pH defines the stability, activity and shelf life parameters. Thus, pharmaceutical formulations are typically formulated with buffers. Various buffers can be used and they are selected to be effective at the desired pH. Exemplary buffers that are often used in almost all existing gonadotrophin formulations are phosphate buffers and citrate buffers. Buffers are required to provide a stable pH to the formulation during its formulation, particularly during its storage, over a range of conditions to which the formulation may be exposed. Finding such a suitable buffer is often very challenging, and then in some cases, an effective buffer can cause undesirable side effects to the formulation, such as pain when citrate buffer is injected. All buffers have inherent disadvantages as additional ingredients in the formulation, which complicates the formulation process, with the risk of adversely affecting other ingredients, stability, shelf life and end user acceptability.

However, the inventors of the present invention unexpectedly provided formulations in the absence of buffering agents without any unacceptable pH fluctuations of the formulation. The formulation comprises arginine as a stabilizing compound. Arginine has an expected buffer capacity of +/-1 pH unit around its pKa, which is 2.17, 12.5 and 9.04. Thus, there is no buffering capacity within the preferred pH range of the present invention (e.g., between 6.0 and 7.5, more preferably between 6.5 and 7.4, preferably at 6.8 or about 6.8). However, it has surprisingly been found that arginine has a strong stabilizing effect on the pH in the specific formulations described in the present invention. This effect of arginine is not described in any field of the prior art.

Advantageously, a mention of such a formulation without the buffer defined in the present invention also provides a formulation with reduced or no pain upon injection and reduced or no skin irritation upon injection. This is in contrast to formulations which use citrate buffers, for example, and which have been shown to cause skin irritation and pain upon injection.

The arginine of the present invention may be arginine, or may be arginine HCl, or even more preferably L-arginine (HCl). The amount of arginine is preferably in the range of 50 to 160mM, more preferably 80 to 160mM, even more preferably about 150mM.

As can be seen from the examples below, arginine has a significant effect on the oxidation of FSH and hCG. However, the inventors do show a particularly high stabilizing effect. It is quite surprising that if gonadotrophin is combined with a highly stable amount of arginine (e.g. 150 mM), they are then able to provide a liquid gonadotrophin formulation that is stable and shows only minimal oxidation (at acceptable levels, see example section below).

This is particularly surprising because the effect is obtained in a pH range away from the pKa value of arginine, i.e. in the range of pH 6.0 to 7.5. This is the preferred pH range of the present invention. In a particularly preferred embodiment, the pH is in the range of 6.5 to 7.4. Even more preferably the pH range is between 6.5 and 7.2. The most preferred pH is 6.8+/-0.3.

Surprisingly, low antioxidant amounts of methionine, preferably L-methionine, can prevent unwanted oxidation. Such low amounts may be as low as 0.05mg/ml, but may be as high as 2.5mg/ml. In a preferred embodiment, the amount of methionine is between 0.1 and 1.5 mg/ml. In a more preferred embodiment, the amount of methionine is between 0.1 and 1 mg/ml. In an even more preferred embodiment, the amount of methionine is between 0.1 and 0.5 mg/ml. The most preferred embodiment is 0.15mg/ml methionine. The high oxidative capacity of arginine can be offset and overcome by such low amounts of methionine, which is not predicted at all.

More unexpectedly, arginine was found to have a more pronounced stabilizing effect than other known stabilizers, such as sucrose, which is a very frequently added and well defined stabilizer.

The formulations may include suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents and/or vehicles. Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil) and injectable organic esters such as ethyl oleate.

In the present invention, especially for multi-dose formulations, it is further preferred to add a preservative. Very preferably the preservative is phenol. In another preferred embodiment, phenol is added at a concentration of 4.0 to 6.0mg/ml, preferably 5.0mg/ml phenol. Surprisingly, phenol is also advantageous in the context of certain inventive formulations compared to other well known preservatives, since in spite of the presence of such preservatives it does not lead to coloration and is very stable, even over a long period of time, as shown in the examples section.

It is further preferred in the present invention to add a surfactant. A very preferred surfactant is polysorbate, even more preferably polysorbate 20. In a further preferred embodiment, polysorbate 20 is added at a concentration of 0.001-0.05mg/ml, preferably 0.005mg/ml polysorbate 20. Advantageously, despite the presence of such a preservative, the formulation of the present invention does not cause turbidity when mixed with polysorbate 20 and is very stable, even over long periods of time, as shown in the examples section.

In addition to the additives already listed above, the composition may also comprise other additives such as, but not limited to, (further) preservatives, wetting agents, emulsifiers and dispersants. Antibacterial and antifungal agents may be included to prevent microbial growth, and include, for example, parabens, chlorobutanol, phenols, sorbic acid, and the like. Furthermore, it may be desirable to include tonicity agents. However, the formulations of the present invention do not contain additional buffering agents.

In some cases, it is desirable to slow down the absorption of gonadotropins such as FSH (and other active ingredients, if present) in subcutaneous or intramuscular injection for prolonged action. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. The absorption rate then depends on the dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of the parenterally administered FSH combination form is achieved by dissolving or suspending the FSH combination in an oily medium.

In accordance with the present invention, the inventors have focused on studying the effect of certain compounds on the stability of liquid gonadotrophin formulations; here, the stabilizing and destabilizing effects of several compounds were investigated. Furthermore, the inventors have attempted to improve the transparency, the degree of coloration, and pain upon injection of the resulting formulation.

The term "stability" may refer to chemical stability, involving covalent modifications in the amino acid sequence, but in the context of protein stability it may also refer to physical stability, involving changes in the folding state (i.e. the natural state) of the protein, excluding covalent bond cleavage. In the present invention, the term "stability" refers to the biostability of gonadotrophin preparations, in particular FSH and hCG. Physical instability of protein formulations may be caused by aggregation of protein molecules to form higher order aggregates, dissociation of heterodimers to monomers, or any other conformational change that reduces at least one biological activity of e.g. FSH proteins (and other active ingredients, if present) included in the present invention.

By "stable" solution or formulation is meant one in which the extent of aggregation, dissociation, conformational modification, loss of biological activity, etc. of the protein is acceptably controlled and does not unacceptably increase over time. Stability may be assessed by methods well known in the art, including measuring light scattering, visual transparency and/or staining, absorbance or optical density, molecular sizing (e.g., by size exclusion chromatography or field flow fractionation), in vitro or in vivo bioactivity, and/or by Differential Scanning Calorimetry (DSC).

The formulations of the present invention are stable for 12 months under storage conditions, preferably 16 months under storage conditions, even more preferably 24 months under storage conditions plus 1 month "in use" (i.e., at room temperature). By stability under "storage conditions" is meant that the formulation is stored in a cooled environment, for example at 5 ℃ ± 3 ℃. In the context of the present invention, "room temperature" means 30℃or below, preferably 15-25℃and preferably 18-25℃and most preferably 25.+ -. 2 ℃.

To determine stability in the context of the present formulation, the well known FSH and hCG (immune) assays are used, which are well known to the person skilled in the art and are described herein only in general terms as follows:

reversed phase high performance liquid chromatography for oxidation(RP-HPLC)

Protein oxidation was determined by RP-HPLC, C4 column gradient elution and UV detection at 210 nm.

Enzyme-linked fluorescence analysis (ELFA)

Immunocompetence of FSH and hCG Using an automatic AnalyzerInstrument (BioMerieux, france) corresponds to FSH and hCG, respectively>The assay is performed in a sandwich immunoassay in a kit.

Size Exclusion Chromatography (SEC)

The purity of rFSH was determined by Size Exclusion Chromatography (SEC) on SEC columns with UV detection at protein ranges 3-70kDa and 210 nm.

Hydrophobic Interaction Chromatography (HIC)

The purity of rhCG was determined by gradient elution on a column with alkylamide groups and UV detection at 220nm using Hydrophobic Interaction Chromatography (HIC).

FSH and LH bioassays

FSH and LH efficacy was determined using an in vivo assay using Steelman-Pohley (FSH) and the seminal vesicle weight gain test (LH), respectively.

Other methods for assessing stability are well known in the art and may also be used in accordance with the present invention.

FSH and LH activity assays were standardized by the world health organization institute of biological standardization (WHO ECBS) using the fourth national standard for urinary FSH and urinary LH at month 11 of 2000 and are well known to those skilled in the art.

It is well known that several preservatives have a remarkable destabilizing effect on gonadotrophin formulations, where it has surprisingly been found that the formulations of the present invention, in particular contain arginine and methionine, but no buffer, in particular stable-even over a long period of time even in the presence of phenol.

The combination of arginine and methionine claimed in the present invention has a stabilizing effect on the liquid hMG formulation which is more pronounced in a more advantageous and surprising manner than the stabilizing effect of known stabilizers such as sucrose. The improved stabilizing effect is particularly surprising compared to known stabilizers such as sucrose. Furthermore, it is highly unexpected that the stabilizing effect of the combination of the invention is still significant despite the absence of buffer.

It is known from the prior art that FSH degradation occurs in pharmaceutical FSH preparations, which has been demonstrated by the examples provided in patent WO 2012/0137442. FSH degrades over time and temperature.

It appears that conformational unfolding of the tertiary and secondary FSH structure occurs upon heating is a two-state transition (when protein aggregation is limited). This expansion may be independent of subunit dissociation (change in quaternary structure).

Furthermore, it is clear in the prior art that FSH containing a preservative such as benzyl alcohol or phenol, which is necessary, for example, as an antimicrobial agent in liquid FSH preparations, clearly has a negative effect on the stability of the FSH multi-dose preparation. Here, the long-term stability of FSH is reduced, the denaturation temperature of FSH is reduced, and the denatured form has a lower level of secondary structure than a preservative-free FSH preparation.

The present invention also relates to a method of stabilizing a liquid gonadotrophin formulation, wherein said method comprises the step of adding arginine and methionine to said formulation, said formulation not comprising a buffer.

Drawings

Fig. 1: titration curves. The slope of the curve shows a buffer capacity, e.g. a steep slope shows a low buffer capacity.

Fig. 2: based on response optimizer results at 25 ℃. The vertical line shows the levels of L-arginine and L-methionine and the response in the FSH and hCG immunoassays is Y (in IU/ml). R is R ² _HCG :92.15% and R ² _FSH ：67.31％。

All studies were confirmed by additional real-time data performed.

Detailed Description

Examples

Example 1

1 background and introduction

Several different pharmaceutical products containing gonadotrophin derived from urine of postmenopausal or pregnant women are used to treat infertility, such as hMG (human menopausal gonadotrophin) formulations. hMG has Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) activity in a one-to-one ratio.

FSH, LH and human chorionic gonadotrophin (hCG) belong to the gonadotrophin family of complex glycoprotein hormones. They are heterodimers consisting of alpha-and beta-subunits. For these Three gonadotrophins, the 92 amino acid α -subunit is common and the β -subunit is unique, giving them different biological properties (Wolfenson C.et al 2005, batch-to-Batch consistency of human-derived gonadotropin preparations compared with recombinant preparations. Reproductive Biomedicine. Vol. 10. No.4:442-454; shen, S.T., cheng, Y.S., shen, T.Y., and Yu, J.Y.2006, molecular cloning of follicle-stimulating hormone (FSH) -beta subunit cDNA from duck pituitary. Gen. Comp endocrinol.148:388-394; fox, K.M., dias, J.A., and Van, R.P.2001, three-dimensional Structure of human follicle-linking hormone mol.endocrinol.15:378-389; burova, T., lecomp, F., galet, C., monsalier, F., delpech, S., haertle, T., and combarnius, Y.2001, conformational stability and in vitro bioactivity of porcine luteinizing horone mol.cell endocrinol.176:129-134 glycoprotein hormone loses biological activity upon dissociation of the noncovalently linked subunits (Alevizaki, M.and Huhtanii, I.2002, structure-function relationships of glycoprotein hormones; lessons from mutations and polymorphisms of the thyrotrophin and gonadotropin subunit genes. Hormones (Athens.) 1:224-232).

LH and hCG bind to the same receptor and thus both have LH activity. In hMG, LH activity is mainly derived from hCG.

The object of the present invention is to develop a gonadotrophin formulation in the form of a liquid formulation for subcutaneous injection. For multi-dose injections, it is often necessary to add preservatives (Meyer, b.k., ni, a., hu, b., and Shi, l.2007, antimicrobial preservative use in parenteral products: past and present.j.pharm.sci.96:3155-3167;Chang,B.S.and Hershenson,S.Practical Approaches to Protein Formulation Development.In Rational Design of Stable Protein Formulations.J.F.Carpenter and M.C.Manning,editors.2002,Plenum Publ., new york.1-25;Pharmaceutical Formulation Development of Peptides and Proteins.2000,CRC Press,Boca Raton).

In general, the natural (bioactive) structure of proteins is very sensitive to its surroundings, such as the composition of the formulation, the container/closure system, the pH and the temperature, and it is a difficulty to determine a suitable liquid formulation of gonadotropins, screening for different excipients. In the work of the present invention, the buffer capacity and real-time stability of various hMG formulations were studied using FSH immunoassay, hCG immunoassay, bioassay, RP-HPLC assay for oxidized protein, titration and pH, as described in detail below.

2 products to be studied

2.1 materials

2.1.1 medicaments (DS)

hMG-HP pharmaceutical (DS) is manufactured by Argentina Instituto Massone S.A., and hMG-HP DS is received as powder by the Feilin pharmaceutical (Ferring Pharmaceuticals) A/S of Copenhagen, denmark.

FSH and LH bioactivity in pharmaceuticals was determined according to the current version of British Pharmacopoeia (BP). The assay can also be performed according to USP version 35 if desired. The biological activity of FSH: LH in hMG is 1:1, so that the average measured biological activity of FSH and LH is used to formulate a pharmaceutical product. Thus, when the concentration of hMG is given as, for example, 625IU/ml, it is equal to the biological activity of 625IU/ml FSH and 625IU/ml LH.

2.1.2 excipients

Table 1 lists the excipients used in this work.

Table 1 list of excipients

2.1.3 Container closure System

For stability studies, the primary packaging materials were glass vials with rubber stoppers and aluminum/plastic caps, or glass cartridges with rubber plungers and crimp caps.

3 manufacturing Process

3.1 mixing

All formulations were manufactured on a laboratory scale.

For the mixing of the drug product solutions (DP), each excipient and the stock solution of Drug (DS) are mixed sequentially. The pH of each formulation was adjusted as necessary before addition of hMG and dilution to final volume. Stock solutions of all excipients and hMG were prepared in Milli-Q water.

3.2 aseptic filtration and aseptic filling

The stable formulation was sterile filtered with a microporous PVDF 0.22 μm filter. Sterile filtration was performed in a LAF bench using an autoclave material.

And (5) filling after filtering. The container is filled with the sample solution. All vials and cartridges were filled under sterile conditions in the LAF bench and immediately closed with rubber stoppers or crimp caps. Outside the LAF bench, the filled vials were sealed with aluminum flip-flops.

4 storage conditions

4.1 storage conditions

The containers containing the pharmaceutical formulation are stored for up to 3 months at 30±2 ℃/65±5% rh under accelerated conditions, and/or for at least 6 months at 25±2 ℃/60±5% rh. At each storage temperature, the container is stored in a vertical position. The cartridge is stored horizontally. All containers were protected from light.

5 analysis method

The analytical methods used in the study are described below.

5.1 titration procedure

As previously described, according to USP-NF, a solution is said to be buffered if it resists changes in ionic activity upon addition of substances that are expected to change ionic activity. A buffer is a substance or combination of substances that imparts such resistance to a solution. Buffer solutions are systems in which ions are in equilibrium with a substance capable of removing or releasing ions. Buffer capacity refers to the amount of material that can be added to a solution without causing a significant change in ionic activity. It is defined as the ratio of added acid or base (in gram-equivalents/liter) to the change in pH units. The capacity of the buffer solution is usually adjusted to the conditions of use by adjusting the concentration of the buffer substance. Buffer capacity is generally expressed as the number of equivalents (Eq) of strong acid or strong base that cause a unit pH change in one liter of the solution, giving the unit mEq/(liter x pH) used to determine the buffer capacity in the present invention. This means that the buffer capacity is defined as the number of moles (equivalents) of H+/OH-required to change the pH of one liter of buffer solution by one unit.

Buffer capacity was determined using a placebo (the placebo refers herein to a formulation without active ingredient) formulation adjusted to the target pH as a starting point. Titration with 0.2N NaOH/HCl was performed to raise or lower the pH. The pH was measured after each addition of 0.2N NaOH/HCl and the volume of 0.2N NaOH/HCl was recorded. The amount of 0.2N NaOH/HCl was plotted as X value and the pH as Y value. For the hMG placebo (target pH), fitting linear regression was performed around pH 6.8, and for the reference placebo (target pH), fitting linear regression was performed around pH 6.5. The buffer capacity can be calculated as μL 0.2N HCl/NaOH used to shift the pH by 0.01pH units/L DP, as well as milliequivalents (mEq) of acid or base/(liter pH units) as described in the definition above.

5.2 FSH and hCG immunoassays

FSH and hCG immunoassays were determined by sandwich ELFA.

5.3 oxidation of proteins

Oxidation of the protein was determined by RP-HPLC.

5.4 pH

pH was measured according to pH.

6. Results

Results of stability study the results of the stability of the buffer evaluation and design of experiment (DoE) study in the buffer capacity study are shown below.

TABLE 2 composition of liquid hMG (600 IU/ml) formulations

1.10 mM disodium hydrogen phosphate dodecahydrate, pH was adjusted with phosphoric acid

2.10 mM trisodium citrate dihydrate, pH adjusted with citric acid

6.1 stability of liquid hMG formulation/3 months

Protein molecules can be stabilized by adding excipients such as salts, carbohydrates or amino acids to the solution, but the degree of stabilization after the addition of different carbohydrates, salts and amino acids varies greatly between different formulations. Here, initial stability studies were performed to screen various combinations of stabilizers with preservatives and buffers in hMG liquid formulations. Surprisingly, the results show that L-arginine has excellent stabilizing effect for hCG immunoassays as shown in tables 3 and 6 and for FSH immunoassays as shown in tables 4 and 7. Formulation E-09 showed excellent stability compared to all other formulations. Formulation E-09 was the only formulation containing L-arginine (see Table 2). Formulations K-01, K-03 and K-04, and K-09 showed excellent stability compared to all other formulations (see Table 6). These formulations also contained L-arginine (see Table 5). In Table 4, formulation E-09 also had the best stability compared to all other formulations for the FSH immunoassay, and Table 7 demonstrates that formulation K-09 shows the highest FSH immunoassay results compared to all other formulations.

Table 3 hCG immunoassays stored at 30±2 ℃/65±5% rh for a period of 1 month. Formulation E-09 showed excellent stability compared to all other formulations. Formulation E-09 contained L-arginine, see Table 2.

(preparations E-04, E-14 and E-15 were excluded from the test due to coloration)

Table 4 FSH immunoassay was performed after 3 months of storage at 30±2 ℃/65±5% rh. Formulation E-09 exhibited the best stability compared to all other formulations. Formulation E-09 contained L-arginine, see Table 2.

6.2 stability of liquid hMG preparation-3 months

Other formulations containing arginine were screened from the initial screen.

TABLE 5 composition stability study of liquid hMG 600 IU/ml-formulation overview

¹ Same as formulation E-09 in Table 2

² Same as formulation E-12 in Table 2

³ Same as formulation E-10 in Table 2

⁴ 10mM disodium hydrogen phosphate dodecahydrate, pH adjusted with phosphoric acid

⁵ 10mM trisodium citrate dihydrate, pH adjusted with citric acid

⁶ Intensity of 530IU/ml

Table 6 hCG immunoassays stored at 25±2 ℃/60±5% rh for a period of 3 months. Formulations K-01, K-03, K-04 and K-09 showed excellent stability compared to all other formulations. Formulations K-01, K-03, K-04 and K-09 contained L-arginine, see Table 5.

It is clear that the formulations comprising L-arginine are much more stable than the formulations comprising different stabilizers. Formulations K-02 and K-03 were compared, for example, after three months.

Table 7 FSH immunoassay during 3 months of storage at 25±2 ℃/60±5% rh. Formulation K-09 showed the highest FSH immunoassay compared to all other formulations. Formulation K-09 contained L-arginine, see Table 5.

The results for hCG were also confirmed for FSH-for example, by comparing preparations K-02 and K-03 after three months.

6.3 buffer Capacity study

Buffers such as sodium phosphate and sodium citrate are physiologically tolerated buffers that are typically added to maintain the pH within the desired range. Trisodium citrate dihydrate and disodium phosphate dodecahydrate were evaluated as buffers by buffer capacity. The buffer capacity was calculated as the amount of acid or base required to shift the pH by a predetermined step, i.e., as described above, the amount of μL 0.2N HCl/NaOH required to shift the pH by 1pH unit/L DP, calculated as mEq acid or base/(liter by pH unit).

Table 8 composition buffer capacity study-formulation overview

1 trisodium citrate dihydrate

2 0.8mM Na ₂ HPO ₄ ×12H ₂ O and about 0.2mM H ₃ PO ₄ Adjusting pH to 6.5

3 reference blank control

4 is equal to 25.3mg/ml L-arginine HCl

The results of titration to determine buffer capacity are shown in figure 1. The slope of the curve represents the buffer capacity, e.g. a steep slope represents a low buffer capacity. As can be seen from FIG. 1, batch C-04 (reference formulation) had the lowest buffer capacity. All of the hMG formulations C-01 (no buffer), C-02 (5 mM trisodium citrate dihydrate) and C-03 (10 mM trisodium citrate dihydrate) had similar buffer capacities at the relevant pH regions of hMG of 6.8.+ -. 0.3, and had higher buffer capacities than the reference formulation containing 1mM disodium hydrogen phosphate dodecahydrate. The target pH of the reference formulation was 6.5. Formulations C-01, C-02 and-03 were compared to a reference formulation known to have a stable pH. Many stability studies of reference formulation C-04 have been performed without any pH drift being observed.

The following table 9 shows the results of the buffer capacity study:

table 9 buffer capacity study. In the pH 6.8 region associated with hMG formulations, all hMG formulations had higher buffer capacity than the reference placebo formulation.

(C-01 and C-04 are mentioned twice because the buffer capacity is calculated in the pH range around two different pH values, respectively)

Surprisingly, the buffer capacity of the hMG formulation without buffer (C-01) was higher than that of the reference formulation C-04. The main excipient component in the hMG preparation is L-arginine at a concentration of 120 mM. The pKa value of L-arginine is pKa1 = 2.17; pKa 2=9.04 and pKa 3=12.5 (handbook of pharmaceutical excipients.2015, pharmaceutical press, london). It is unexpected that arginine exhibits sufficient buffering capacity away from pKa values.

6.4 Oxidation Studies

After 3 months of storage at 30.+ -. 2 ℃ C./65.+ -. 5% RH, formulation E-09 (see Table 5) containing L-arginine increased the oxidized protein level by 241% relative to the initial value (see Table 10 below). This is a very high amount and in some cases such a high amount of oxidation is undesirable.

Therefore, further studies have been made to investigate whether this problem can be overcome.

There are several alternative antioxidants available for protein formulations. Methionine may be added to prevent oxidation by a mechanism that competes with the oxidation of methionine residues in the protein. The results of methionine addition to the arginine-containing formulations are shown in Table 10. Formulations K-01, K-03, K-04 and K-09 contain arginine and methionine, and the level of oxidized protein is significantly and advantageously reduced. Formulation K-01 (with methionine) was identical to formulation E-09 (without methionine). Comparing the two batches, it can be seen that methionine greatly reduces the level of oxidized protein.

Table 10% oxidized protein compared to initial increase during 3 months of storage at 30±2 ℃/65±5% rh. Formulation E-09 contained arginine and no methionine. Formulations K-01, K-03, K-04 and K-09 contained arginine and methionine, see Table 5.

(preparations E-04, E-14 and E-15 are excluded due to coloring)

Taken together, these two studies indicate that arginine has a great stabilizing effect and that even small amounts of methionine have an antioxidant effect in gonadotrophin formulations.

6.5 DoE stability study

In combination with the results of the above studies and buffer capacity studies, doE studies were performed to study the interaction between arginine and methionine and to determine the optimal concentration of these two excipients.

Table 11 composition-formulation overview for DoE stability study of liquid hMG 600IU/ml

¹ Trisodium citrate dihydrate

² For formulations without buffer, the pH was adjusted with 0.2N HCl/NaOH;

³ for trisodium citrate containing dihydrateFormulation of compound buffer the adjustment of pH with 0.2N NaOH/0.5M citric acid monohydrate includes as reference formulation

6.5.1 FSH immunoassay

The stability results of the FSH immunoassay during storage at 25±2 ℃/60±5% rh for 3 months are listed in table 12.

Table 12 results of FSH immunoassay during storage at 25±2 ℃/60±5% rh. Table 11 lists a complete description of all formulations.

A statistical calculation was performed on FSH activity [ IU/ml ] to assess the effect and interaction of arginine and methionine. The results of the statistical evaluation confirm that L-arginine has a statistically significant effect on the stability results of FSH immunoassays. Methionine has no significant statistical impact on FSH immunoassay stability results. For this response parameter, there was no statistically significant interaction between arginine and methionine.

6.5.2 hCG immunoassay

The stability results of hCG immunoassays during storage at 25±2 ℃/60±5% rh for 3 months are listed in table 13.

Table 13 results of hCG immunoassay during storage at 25±2 ℃/60±5% rh. Table 11 lists a complete description of all formulations.

Statistical calculations were performed on hCG activity [ IU/ml ] to assess the effect and interaction of arginine and methionine. The results of the statistical evaluation confirm that arginine has a statistically significant effect on hCG immunoassay stability results. Methionine content had less effect on hCG immunoassay stability results at 25 ℃. It is clear that at low (0.1 mg/ml) methionine concentrations, stability is not further improved by increasing the concentration even up to 1.5 mg/ml. For this response parameter, there was no statistically significant interaction between arginine and methionine.

6.5.3 oxidation of proteins

In the above stability studies, it was surprisingly observed that the presence of arginine has a very large stabilizing effect. However, the addition of arginine resulted in increased levels of oxidized protein. Methionine may be added to prevent this oxidation and the present study examined the concentration balance between arginine as a stabilizer and methionine as an antioxidant.

The stability results of oxidized protein amounts [ relative to the initial increase% ] for formulations containing arginine and methionine (concentration range 0.1-1.5 mg/ml) during 6 months of storage at 25.+ -. 2 ℃ C./60.+ -. 5% RH and during 3 months of storage at 30.+ -. 2 ℃ C./65.+ -. 5% RH are listed below. For comparison, table 14 also includes formulation E-09, which contains arginine but not methionine.

Table 14 shows the results of oxidizing proteins during storage at 25.+ -. 2 ℃/60.+ -. 5% RH and 30.+ -. 2 ℃/65.+ -. 5% RH.

Table 11 lists a complete description of all formulations.

It can be seen unexpectedly from Table 14 that even small amounts of methionine are sufficient to prevent oxidation of arginine over the entire concentration range.

6.5.4 DoE overview

To arrive at and summarize the DoE results, a response optimizer in Minitab was applied, see fig. 2. The Minitab response optimizer shows how different experimental factor settings, e.g. arginine and methionine concentrations, affect the predicted responses, e.g. factor engineered FSH immunoassay [ IU/ml ] and hCG immunoassay [ IU/ml ]. The optimization graph shows the effect of each factor (column) on the response (row). The vertical thick line on the graph represents the current factor setting. The numbers shown at the top of the column show the current factor level setting (in brackets). The horizontal dashed line and corresponding numbers represent the response of the current factor level. The response optimizer will be based on the 25 ℃ results. The results of applying the response optimizer are shown in fig. 2. Optimal stability (combined expected) is achieved at high concentrations of arginine and low concentrations of methionine.

6.6 pH study

In the buffer capacity study, no buffer was shown to be needed in hMG formulations, and only arginine was used to stabilize the pH within the desired pH range.

To confirm that the pH remained unchanged during storage, the pH was measured for 6 months at 25.+ -. 2 ℃ C./60.+ -. 5% RH and 3 months at 30.+ -. 2 ℃ C./65.+ -. 5% RH. The results are shown in Table 15.

The target pH for all formulations was pH 6.8, and at the initial time point the pH for all formulations was 6.8. The pH was fairly stable but increased slightly to about 6.9 after 2 months of storage and maintained at a pH of about 6.9 to 6 months of storage. The storage of the results for up to 6 months confirms the stability of the pH over the tested concentration range of arginine.

Table 15 PH study results

Conclusion 7

Very surprisingly, the buffer capacity of the hMG formulation without buffer was higher than the reference formulation containing 1mM disodium hydrogen phosphate dodecahydrate. The main excipient component in hMG formulations is arginine. Arginine has a pKa value of pKa ₁ ＝2.17；pKa ₂ ＝9.04，pKa ₃ =12.5. It is unexpected that arginine exhibits sufficient buffering behavior away from its pKa value. However, the results clearly show that no additional buffer is needed in the hMG formulation in the current pH range. Arginine alone is sufficient to maintain the pH within the desired pH range. The DoE study demonstrates the stability of pH, which tests the concentration range of arginine stored for 6 months at 25±2 ℃/60±5% rh and 3 months at 30±2 ℃/65±5% rh, which demonstrates the observations in the buffer capacity study.

The addition of arginine to the liquid hMG formulations of the invention showed a great stabilizing effect on the liquid hMG formulation. However, arginine has also been found to result in high levels of oxidized protein. The addition of methionine was shown to prevent oxidation compared to formulations without methionine.

Storage for 6 months at 25.+ -. 2 ℃/60.+ -. 5% RH and 3 months at 30.+ -. 2 ℃/65.+ -. 5% RH showed that even small concentrations of methionine prevented oxidation.

From a summary of the results of these studies, it is clear that the addition of arginine is sufficient to maintain the pH at the desired pH level. The addition of arginine greatly stabilizes the gonadotrophin formulation being tested in the liquid formulation. No other amino acids, sugars or salts tested showed similar stabilizing effects. By adding arginine the level of oxidized protein is significantly increased, whereas the addition of even small amounts of methionine prevents oxidation of the protein. Surprisingly, even small amounts of methionine are sufficient to prevent oxidation, irrespective of the arginine concentration.

Example 2

The inventors have further shown that the advantageous inventive formulations of the present invention are also suitable for stabilizing corresponding recombinant gonadotropin formulations.

For this purpose, recombinant FSH and recombinant hCG (each having the above sequence) are prepared according to well-known methods.

As described below, accelerated stability studies were performed on both formulations, including rhCG or rFSH, respectively.

The surprising stabilization of arginine observed was also demonstrated in recombinant proteins. Both rFSH and rhCG were formulated at 5mg/ml phenol, 0.15mg/ml L-methionine, 150mM arginine HCl, 0.005mg/ml polysorbate 20, pH 6.8. To simplify the analysis of protein stability, rFSH and rhCG were formulated in different containers.

Table 16

This is a good demonstration that the formulations of the invention containing arginine, but no additional buffer, and a small amount of methionine are also suitable for use with stable recombinant gonadotropins.

Example 3

Further studies were performed in which the following compositions were evaluated:

TABLE 17 composition of liquid hMG 625IU/ml formulation

¹ pH adjustment with 0.2N HCl/NaOH

Stability of the target formulation

In combination with all previous results, good stability of the above formulation was confirmed as follows:

table 18 FSH immunoassay

FSH immunoassay stability results stored for 3 months at 25±2 ℃/60±5% Relative Humidity (RH) and 12 months at 5±3 ℃):

FSH immunoassay results during storage at 25±2 ℃/60±5% rh and during storage at 5±3 ℃ for 12 months

The results confirm the stability of the FSH immunoassay assay.

Table 19 hCG immunoassay

The stability results of hCG immunoassays stored for 1 month at 25±2 ℃/60±5% rh and 12 months at 5±3 ℃ are listed in table 19.

hCG immunoassay results during storage at 25±2 ℃/60±5% rh and during 12 months at 5±3 ℃

The results confirm the stability of hCG immunoassay assay.

TABLE 20 oxidized proteins

Stability results of the amount of oxidized protein [ relative to% initial increase ] during storage at 25±2 ℃/60±5% rh for 3 months and storage at 5±3 ℃ for 12 months.

Results of oxidation of proteins during storage at 25.+ -. 2 ℃/60.+ -. 5% RH and during 12 months at 5.+ -. 3 ℃

pH

The pH stability results during storage at 25±2 ℃/60±5% rh for 3 months and storage at 5±3 ℃ for 12 months showed stable pH.

Table 21 pH results during 25.+ -. 2 ℃/60.+ -. 5% RH storage and during 12 months at 5.+ -. 3 ℃

Example 4

In addition to the above, the inventors performed a bioassay study (for DoE batches, stored at 25 ℃ for 6 months).

FSH bioassay (Steelman-Pohley)

Table 22 lists the stability results of FSH bioassays during 6 months of storage at 25±2 ℃/60±5% rh.

Table 22 results of FSH bioassays during storage at 25±2 ℃/60±5% rh. A complete description of all formulations is given in table 11.

The results confirm the stability of the FSH bioassay assay.

LH bioassay (semen weight gain)

The stability results of the LH bioassays during 6 months of storage at 25.+ -. 2 ℃/60.+ -. 5% RH are shown in Table 23.

Table 23 results of LH bioassays during storage at 25.+ -. 2 ℃/60.+ -. 5% RH. A complete description of all formulations is given in table 11.

The results confirm the stability of the LH bioassay assay.

In addition, the composition of example 3 was also tested in FSH and LH bioassays, with the following results:

table 24 lists the stability results of FSH bioassays during storage at 25±2 ℃/60±5% rh for 3 months and at 5±3 ℃ for 12 months:

table 24 results of FSH bioassays during storage at 25±2 ℃/60±5% rh and during 12 months at 5±3 ℃.

The results confirm the stability as determined by FSH bioassay.

Stability results of LH bioassays during storage at 25±2 ℃/60±5% rh for 3 months and at 5±3 ℃ for 12 months are listed in table 25.

Table 25 results of LH bioassays during storage at 25.+ -. 2 ℃/60.+ -. 5% RH and during 12 months at 5.+ -. 3 ℃.

The results confirm the stability of the LH bioassay assay.

8 abbreviations and definitions

BP British pharmacopoeia

Design of DoE experiment

DS medicine

FSH follicle stimulating hormone

hCG human chorionic gonadotrophin

hMG human menopausal gonadotropin

hMG-HP human menopausal gonadotrophin-high purification

JP Japanese pharmacopoeia

LH luteinizing hormone

MD multi-dose

NF American national drug Collection

Ph Eur European Pharmacopeia

PS 20 Polysorbate 20

USP united states pharmacopoeia

WFI (Water-Fidelity) injection water

Sequence listing

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Claims

1. A liquid gonadotrophin pharmaceutical formulation consisting of the following components:

gonadotrophin

-50-160mM arginine HCl

0.05 to 1.5mg/ml of L-methionine

-0.001-0.05mg/ml polysorbate 20

4.0 to 6.0mg/ml phenol

pH 6.0 to 7.5, pH referring to the pH of the whole solution

Water for injection (WFI).

2. A pharmaceutical formulation according to claim 1, wherein the gonadotropin comprises hCG (human chorionic gonadotrophin), and optionally FSH and/or LH.

3. The pharmaceutical formulation according to claim 1 or 2, wherein the gonadotrophin comprises hMG (human menopausal gonadotrophin).

4. A pharmaceutical formulation according to claim 3, wherein hMG is present in the formulation in an amount of 35-850IU/ml.

5. A pharmaceutical formulation according to claim 3, wherein hMG is present in the formulation in an amount of 50-800IU/ml.

6. A pharmaceutical formulation according to claim 3, wherein hMG is present in the formulation in an amount of 100-700IU/ml.

7. A pharmaceutical formulation according to claim 3, wherein hMG is present in the formulation in an amount of 625IU/ml.

8. The pharmaceutical formulation according to any one of claims 1-7, wherein the arginine HCl is present in the formulation in an amount of 150mM.

9. The pharmaceutical formulation according to any one of claims 1 to 7, wherein L-methionine is present in the formulation in an amount of 0.15mg/ml.

10. The pharmaceutical formulation according to any one of claims 1-7, wherein polysorbate 20 is present in the formulation in an amount of 0.005mg/ml.

11. The pharmaceutical formulation according to any one of claims 1 to 7, wherein the phenol is present in the formulation in an amount of 5.0mg/ml.

12. The pharmaceutical formulation according to any one of claims 1-7, wherein the pH is 6.8+/-0.3.

13. Use of a liquid pharmaceutical formulation according to any one of claims 1-12 in the manufacture of a medicament for the treatment of infertility.

14. Use according to claim 13, characterized in that it is for Ovulation Induction (OI), assisted Reproductive Technology (ART) and/or male hypogonadism.